Fig. 8.
Fig. 8. AP-1 is not crucial for the activation of9E3/cCAF in normal fibroblasts. / The transfection conditions were described in Figure 2B and Figure 5. (A) Overexpression of the AP-1 complex (c-Fos andc-Jun) to determine its functionality in primary normal fibroblasts. A heterologous promoter system containing 7 AP-1 binding elements in series in front of the luciferase reporter gene (Cis-Path Detect System from Stratagene) was used for these experiments. Cells transfected with pFR-AP1 showed that in the context of the tandem of AP-1 binding sites, thrombin can stimulate AP-1–dependent transcription (ii). Overexpression of c-Fosand c-Jun by cotransfection of 1 μg of each plasmid and 2 μg of pFR-AP-1 in the absence (iii) or presence (iv) of thrombin resulted in a 6- and 11-fold increase, respectively, in transcription of the reporter system. MEK kinase (MEKK) overexpression was used as a positive control for the system; cells cotransfected with 2 μg of pFR-AP-1 and 1 μg of the MEKK expression vector showed a high activation of the reporter gene (v). (B) Activation of the AP-1 binding element in the context of the 9E3/cCAF promoter. The fibroblasts were cotransfected with 2 μg of p683 (i-vi) or pmElk1 (vii-xii) and 1 μg of the expression vectors forc-Fos and c-Jun. Transfections with p683,c-Fos, and c-Jun individually or in combination did not stimulate expression of the reporter gene above that of the control (iii,iv,v); overexpression of c-Fos andc-Jun had no effect on stimulation by thrombin (compare ii and vi). With the mutated Elk1 construct (pmElk1), there was no enhancement of reporter transcription with thrombin stimulation (viii), AP-1 component(s) overexpression (ix,x,xi) or their combination (xii). (C) Mutation of the AP-1 binding element within the context of the p683 promoter. An insignificant reduction in reporter activation by pmAP-1 was observed when compared to the p683 control. DMSO, the vehicle for the PD98059 inhibitor, stimulated a small increase (2 ×) for both the mutated and nonmutated p683. PD98059, a highly selective inhibitor of MEK1, abrogated thrombin stimulation of the reporter. For each experiment, representative data are shown in which all conditions were performed simultaneously in the same batch of cells. Each type of experiment was performed at least twice, using different batches of cells. Bars represent SEMs of 3 samples per condition.

AP-1 is not crucial for the activation of9E3/cCAF in normal fibroblasts.

The transfection conditions were described in Figure 2B and Figure 5. (A) Overexpression of the AP-1 complex (c-Fos andc-Jun) to determine its functionality in primary normal fibroblasts. A heterologous promoter system containing 7 AP-1 binding elements in series in front of the luciferase reporter gene (Cis-Path Detect System from Stratagene) was used for these experiments. Cells transfected with pFR-AP1 showed that in the context of the tandem of AP-1 binding sites, thrombin can stimulate AP-1–dependent transcription (ii). Overexpression of c-Fosand c-Jun by cotransfection of 1 μg of each plasmid and 2 μg of pFR-AP-1 in the absence (iii) or presence (iv) of thrombin resulted in a 6- and 11-fold increase, respectively, in transcription of the reporter system. MEK kinase (MEKK) overexpression was used as a positive control for the system; cells cotransfected with 2 μg of pFR-AP-1 and 1 μg of the MEKK expression vector showed a high activation of the reporter gene (v). (B) Activation of the AP-1 binding element in the context of the 9E3/cCAF promoter. The fibroblasts were cotransfected with 2 μg of p683 (i-vi) or pmElk1 (vii-xii) and 1 μg of the expression vectors forc-Fos and c-Jun. Transfections with p683,c-Fos, and c-Jun individually or in combination did not stimulate expression of the reporter gene above that of the control (iii,iv,v); overexpression of c-Fos andc-Jun had no effect on stimulation by thrombin (compare ii and vi). With the mutated Elk1 construct (pmElk1), there was no enhancement of reporter transcription with thrombin stimulation (viii), AP-1 component(s) overexpression (ix,x,xi) or their combination (xii). (C) Mutation of the AP-1 binding element within the context of the p683 promoter. An insignificant reduction in reporter activation by pmAP-1 was observed when compared to the p683 control. DMSO, the vehicle for the PD98059 inhibitor, stimulated a small increase (2 ×) for both the mutated and nonmutated p683. PD98059, a highly selective inhibitor of MEK1, abrogated thrombin stimulation of the reporter. For each experiment, representative data are shown in which all conditions were performed simultaneously in the same batch of cells. Each type of experiment was performed at least twice, using different batches of cells. Bars represent SEMs of 3 samples per condition.

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